The ubiquitous distribution of glycoconjugates at cell surfaces, extracellular matrices and within particular organelles has focused a great deal of research on the study of glycoconjugates as molecular determinants in cellular function, intracellular processing and intracellular interactions. Oligosaccharides may be covalently linked to the protein through an asparagine side chain (N-linked) or through a serine or threonine side chain (O-linked).
Differential glycosylation of a polypeptide can create different subsets of glycoproteins which may have different physical and biochemical properties [Rademacher, T. W., Parekh, R. B., and Dwek, R. A. (1988) Annual Review Biochemistry 57, 785-838]. This may result in functional diversity leading to different disease states. For example, there is a shift in the population of the glycosylated forms of IgG towards those with a higher content of agalactosyl biantennary N-linked oligosaccharides in active rheumatoid arthritis, tuberculosis and Crohn's disease. This shift is thought to be involved in disease pathogenesis [Parekh, R. B., Dwek, R. A., Sutton, B. J. et al (1985) Nature 316, 452-457].
One known method of analysis of glycoproteins involves the following steps:
i) controlled hydrazinolysis of the glycoprotein to release intact oligosaccharide moieties or digestion with a glycopeptidase, PA1 ii) labelling with tritium in a reduction reaction, PA1 iii) digestion of the oligosaccharides with specific glycosidases, and PA1 iv) fractionation of the resulting products by chromatography. PA1 a) a cavity layer of dielectric material of refractive index n.sub.3, PA1 b) a dielectric substrate of refractive index n.sub.1, and PA1 c) interposed between the cavity layer and the substrate, a dielectric spacer layer of refractive index n.sub.2.
The fractionation profile is compared to the elution parameters of standard oligosaccharide moieties to give a compositional analysis. The oligosaccharide moieties may also be released from the glycoprotein using enzymes such as endoglycosidase H.
An alternative strategy based on fast atom bombardment mass spectroscopy for analysis of glycoproteins has also been developed [Dell, A., Advances in Carbohydrate Chemistry and Biochemistry 45, 19-72].
These methods are time-consuming, and involve the use of radioactivity and/or sophisticated equipment.
We have now devised methods and apparatus for glycosylation analysis which overcome or substantially mitigate certain of the above-mentioned disadvantages, and are particularly useful when very detailed analysis of oligosaccharide composition is not required.